EP2046814A2 - Petits peptides minimisés présentant une forte affinité au facteur viii et protéines similaires au facteur viii - Google Patents

Petits peptides minimisés présentant une forte affinité au facteur viii et protéines similaires au facteur viii

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Publication number
EP2046814A2
EP2046814A2 EP07787022A EP07787022A EP2046814A2 EP 2046814 A2 EP2046814 A2 EP 2046814A2 EP 07787022 A EP07787022 A EP 07787022A EP 07787022 A EP07787022 A EP 07787022A EP 2046814 A2 EP2046814 A2 EP 2046814A2
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EP
European Patent Office
Prior art keywords
fviii
compound
bpa
domains
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP07787022A
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German (de)
English (en)
Inventor
Sebastian Knör
Horst Kessler
Charlotte Hauser
Evgueni Saenko
Alexey Khrenov
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Technische Universitaet Muenchen
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Technische Universitaet Muenchen
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Priority to EP07787022A priority Critical patent/EP2046814A2/fr
Publication of EP2046814A2 publication Critical patent/EP2046814A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)

Definitions

  • the present invention is related to the composition of small molecules and their use in the field of protein isolation, purification, stabilizing and/or enhancing its activity.
  • the present invention relates to the synthesis and optimization of compounds comprising small peptides and peptide derivatives with affinity to coagulation Factor VIII and/or Factor VHI-like polypeptides and/or domains thereof.
  • These compounds are useful for labeling, detecting, identifying, isolating and preferably for purifying, stabilizing and enhancing the activity of Factor VIII, Factor Vlll-like polypeptides or domains thereof from physiological and non-physiological solutions comprising same.
  • these compounds may be used as ligands. which bind Factor VIII, Factor Vlll-like polypeptides or domains thereof in methods of the present invention.
  • Factor VIII is an essential component of the intrinsic pathway of biood coagulation (Bolton-Maggs, P. H. B.; K, J. Pasi Lancet 2003, 361. 1801 ). This plasma protein is circulating in blood in complex with von Willebrand factor (vWf), which protects and stabilizes it. Genetic deficiency of FVIII function results in a life-threatening bleeding disorder known as Hemophilia A, one of the most common bleeding disorders, which is treated by repeated infusions of FVIII. Hemophilia A is the result of an inherited deficiency of Factor VIII. For medical treatment, patients are given doses of Factor VIII derived from either blood plasma or recombinant cells.
  • Hemophilia A the hereditary X chromosome-linked bleeding disorder caused by deficiency or structural defects in a coagulation Factor VIII (FVIII) 5 affects approximately one in 5000 males.
  • the clinical severity of Hemophilia A correlates with the degree of factor deficiency and is classified as severe disease with FV ⁇ II levels of less than 1%, moderate (1-5% FVIII levels) and mild disease ( 5-25% FVIII levels).
  • the disease is characterized by spontaneous bleedings, as well as by uncontrollable bleedings in case of trauma or surgery.
  • Hemophilia A Other clinical hallmarks of Hemophilia A are acute recurrent painful hemarthrosis, which can progress to chronic arthropathy characterized by progressive destruction of the cartilage and the adjacent bone, muscle hematoma, intracerebral hemorrhages and hematuria (Klinge, J.; Ananyeva, N. M.; Hauser, C. A.; Saenko, E. L. Semin. Thromb. Hemost. 2002, 28, 309-322).
  • Hemophilia A is treated by repeated infusions of FVIII 1 derived from either human blood plasma or recombinant cells, expressing FVIII.
  • the FVIII molecule (-300 kDa, 2332 amino acid residues) consists of three homologous A domains, two homologous C domains and the unique B domain, which are arranged in the order of A1-A2-B-A3-C1-C2.
  • FVIII Prior to its secretion into plasma, FVIII is processed intracellularly to a Me 2+ -linked heterodimer produced by cleavage at the B-A3 junction. This cleavage generates the heavy chain (HCh) consisting of the Al (1-372), A2 (373-740) and B domains (741-1648) and the light chain (LCh) composed of the A3 (1690-2019), Cl (2020-2172) and C2 (2173- 2332) domains.
  • HCh heavy chain
  • LCh light chain
  • the resulting protein is heterologous in size due to a number of additional cleavages within the B domain, giving the molecules with B-domains of different length.
  • the C-terminal portions of the Al (amino acids 337-372) and A2 (amino acids 711-740) domains and the /V-terminal portion of LCh (amino acids 1649-1689) contain a high number of negatively charged residues and are called acidic regions (ARl, AR2 and AR3, respectively).
  • KOGENATE FS (Bayer) has been developed as a second generation product. Different to KOGENATE, KOGENATE FS is cultured in cell culture medium containing recombinant insulin and Human Plasma Protein Solution (HPPS), but no proteins derived from animal sources.
  • HPPS Human Plasma Protein Solution
  • REFACTO Wired-Ayerst Pharmacia and Upjohn
  • BDDrFVIII the first licensed B domain deleted recombinant FVIII molecule
  • the r-FVIII SQ gene which encodes a single chain 170 kDa polypeptide, was derived from full-length cDNA by removing the major part of the region encoding the B-domain.
  • the r-FVIll SQ vector system was inserted into CHO cells and cultured in a serum-free medium supplemented with human albumin and recombinant insulin.
  • the purification comprises five different chromatography steps including immunoaffinity with monoclonal antibodies directed to the heavy chain of FVIIl, and a chemical solvent/detergent virus inactivation step (Eriksson, R. K.; Ferige, C; Lmdner- Olsson, E.; Ljungqvist, C; Rosenquist, J.; Sraeds, A. L.; Ostlin, A.; Charlebois, T,; Leonard, M-; Kelley, B. D.; Ljungqvist, A. Sem, Hematol. 2001, 38, 24-31).
  • FVIII products It is important in the development of FVIII products to avoid any use of animal or human proteins in order to improve safety. In contrast to currently licensed recombinant FVIII preparations, next generation FVIII products will adapt production methods to culture media that do not contain any components of human or animal origin. Thus, the ultimate goal is an improvement in the production of FVIII to the point of completely avoiding any contact with components derived from animal or human raw materials. There is also a demand to improve purification methods for FVIIl. Methods offering FV ⁇ II of high purity and activity obtained directly from various solutions such as blood or cell culture supernatants remain in demand, thereby, reducing the number of purification steps, and cost involved. New methods to gain FVIII in a faster, more efficient and cost-effective way remain unrealized by the current art.
  • Factor VIII is usually concentrated by affinity chromatography, employing monoclonal antibodies as ligands (Amatschek, K; Necina, R.; Hahn, R.; Schallaun, E.; Schwinn, H.; Josic, D.; Jungbauer, A. J. High Resol. Chromatogr. 2000, 23, 47-58).
  • oligo- and polypeptides as the partners of affinity ligands polypeptides has been suggested (see, WO 99/14232; or US 2003-165822, each incorporated herein by reference in their entirety). Nevertheless, this method still has disadvantages.
  • the large scale synthesis and purification of the mentioned oligopeptides is not trivial and quiet cost- intensive.
  • these oligopeptides are sensitive towards proteolytic degradation and the presence of proteases cannot be completely avoided if raw materials derived from blood or cell cultures are applied to the affinity column. This may rapidly lead to inefficiency and reduced selectivity of the affinity purification step and, furthermore, to a reduced purity and half life of the eluted factor samples as well as half life of the expensive column material.
  • the present invention is directed to a next generation product that is produced in culture media devoid of any components of human or animal origin using compounds and methods described herein. Furthermore, the present invention may also be directed to purification of plasma-derived FVIII preparations.
  • the present invention includes compounds comprising chemically synthesized unique high- affinity peptides and peptide derivatives which can replace monoclonal antibodies and have improved proteolytic stability compared to the known oligopeptides mentioned above. Furthermore, the inventive compounds are suitable for large scale solution synthesis and therefore will minimize the production costs.
  • the present invention is directed to specific compounds comprising peptides and peptide derivatives. These compounds exhibit particular properties of binding and/or releasing FVIII or FVIII-like polypeptides or domains thereof, wherein the domains are substantially as defined above for the FVII ⁇ -molecule, and may serve as ligands for affinity separation of FVIII, FVIII-like polypeptides or domains thereof.
  • the compounds of the present invention comprising peptides and peptide derivatives are tetrapeptides, pentapeptides, hexapeptides and heptapeptides that bind FVIII or FVIII-like proteins or domains thereof with affinity sufficient for chromatographic purification of FVIII.
  • the compounds are binding molecules that exhibit distinct characteristics for binding of the target Factor VIII polypeptides as well as specific characteristics for release (elution) of the target polypeptides (i.e. specific composition and pH of application and elution buffers).
  • specific characteristics for release (elution) of the target polypeptides i.e. specific composition and pH of application and elution buffers.
  • the compounds may easily be modified by existing chemical methods. Such modification is not technically feasible for the conventionally used antibodies.
  • a further embodiment relates to an inert matrix as solid carrier material comprising the immobilized compound, preferably a peptide or peptide derivative.
  • the solid carrier material is a polymeric material.
  • the compound is chemically bound to the solid carrier matrix.
  • the compound is chemically bound to the solid carrier matrix via an anchoring molecule.
  • the compound is chemically bound to the solid carrier matrix via a spacer molecule. It is also contemplated that the compound is chemically bound to the solid carrier matrix through an anchoring molecule and an additional spacer molecule.
  • the present invention relates to a diagnostic device or kit comprising a compound of the present invention immobilized on a matrix, wherein the compound binds specifically to a FVIII or FVIII-related protein or domains thereof.
  • the compound is directly or via an anchoring compound and/or a spacer molecule immobilized on the matrix, which may be a polymeric material such as, for example, a resin,
  • the compounds are used in methods as a label of a FVIII or FVIII -like protein or domains thereof.
  • the compound is used in methods of identification and/or purification of FVIII or FVIII-like proteins or domains thereof.
  • the compound in another embodiment, is used in methods to stabilize FVI ⁇ I or FVIII-like proteins or domains thereof, substantially as defined above for the FVIII- mo ⁇ ecuie.
  • the compound is used in methods to enhance the activity of FVIII or FVIII-like proteins.
  • the present invention relates further to the medical use of the compound of the present invention in the treatment of diseases. DESCRIPTION OF THE FIGURE
  • Figure 1 depicts the structures of compounds S7 and S8 according to the present invention.
  • the affinity chromatography is a well established powerful technique which is a state-of-the- art procedure used for purification of complex molecules such as proteins (Jack, G, W,; Beer, D. J. Methods MoL Biol. 1996, 59, 187-196).
  • Affinity chromatography offers the unique possibility to isolate the target protein with excellent selectivity from contaminating proteins by its strong interaction between a target molecule and a ligand, which is immobilized on a resin.
  • the ligands are either polyclonal or monoclonal antibodies. Monoclonal antibodies are preferred, because they are monospecific and can be produced with precision (Scopes, R, K, Protein purification: Principles and Practice. Springer, New York, 1994).
  • FVIII is a large and complex protein which plays an important function in the blood coagulation cascade and has great therapeutic significance. Deficiencies in FVIH production in vivo caused by genetic mutations can lead to hemophilia which is treated by infusion of purified preparations of human FVIII (Lee, C. Thromb. Haemosi. 1999, 82, 516-524).
  • the current sources of human FVIII for treatment of hemophilic patients are plasma-derived FVI ⁇ I and recombinant FVIII, the latter synthesized in Chinese hamster ovary (CHO) cells (Kaufman, R. J.; Wasley, L. C; Dorner, A. J. J. Biol Chem. 1988, 263, 6352-6362) and baby hamster kidney (BHK) cells (Boedeker, B. D. G. Sent. Thromb. Hemost. 2001, 27, 385-394). In addition to high purity criteria, it is critical to ensure immunological and virus safety.
  • the use of an immunoaffinity chromatography resin is a common manufacturing procedure for all recombinant FVIII preparations, and for many plasma-derived FVIII products.
  • the current manufacturing process which includes affinity chromatography uses a monoclonal antibody (mAb) that is specific for FVIII (Lee, C, Recombinant clotting factors in the treatment of hemophilia. Thromb, Haemost, 1999, 82, 516-524).
  • mAb monoclonal antibody
  • the current industrial FVIII purification utilizes a mAb immunoaffinity step providing excellent removal of process-related impurities such as DNA and host cells.
  • the present invention fulfills this need by providing a compound which is a chemically synthesized small peptide in an immunoaffinity chromatography purification method that offers a reduction or elimination of several of the described pitfalls.
  • the compounds of this invention comprising small peptides and peptide derivatives have advantages as ligands, because they are unlikely to provoke immune responses in case of leakage into the product. Small peptides and peptide derivatives are also much more stable in comparison with antibodies. Another advantage is their significant lower production costs, since they can easily be manufactured aseptically in huge quantities under good manufacturing practices (GMP).
  • GMP good manufacturing practices
  • the use of the small peptides and peptide derivatives and methods of the present invention achieve a purified product using no animal-derived or human-derived raw materials. Last but not least, the sophisticated chemical synthesis described herein allows refined steps to improve the affinity of the small peptides and peptide derivatives towards their target protein.
  • the present invention provides the ordinary artisan working in the field with a compound and a process that improves the commoniy used purification procedure of FVIII.
  • the present invention provides a FVIII purification method that avoids the use of mouse monoclonal antibodies for immunoaffinity purification of FVIII.
  • the invention includes chemically synthesized unique high-affinity peptides and peptide derivatives which can replace monoclonal antibodies and have improved proteolytic stability compared to the known oligopeptides mentioned above. This would meet the up-to-date requirements for biological safely.
  • the inventive compounds are suitable for large scale solution synthesis and therefore minimize the production costs of the affinity ligands.
  • the present invention comprises novel compounds, preferably pentapeptides and hexapeptides and derivatives thereof as ligands for detecting, identifying, isolating and purifying as well as labeling active Factor VIII, Factor VIII-Hke proteins or domains thereof from solutions that contain such proteins.
  • the Factor VIII binding molecules of the present invention exhibit remarkable stability as well as high affinity for Factor VIII, Factor V ⁇ II-like peptides or domains thereof.
  • the invention provides a cost-effective means to ensure fast separation and purification of commercial quantities of proteins useful in the treatment of hemophilia A.
  • the terms “Factor V ⁇ II and Factor VIII- like proteins” encompass any Factor VIII protein molecule from any animal, any recombinant or hybrid Factor VIII or any modified Factor VIII.
  • such "Factor VIII and Factor Vlll-like proteins” are characterized by an activity (as determined by the standard one stage clotting assay, as described e.g., in Bowie, E. J. W., and C. A. Owen, in Disorders of Hemostasis (Ratnoff and Forbes, eds.) pp. 43-72. Gr ⁇ nn & Stratton, Inc., Orlando, FIa. (1984)), of at least 10%, more preferably at least 50%, most preferably at least 80%, of the activity of native human form of Factor VIII.
  • Factor Vlll-like proteins also encompass domains, fragments and epitopes of factor VIII proteins of any source, as well as hybrid combinations thereof.
  • the term "Factor Vlll-iike proteins” furthermore includes fragments of Factor VIII, which can be used as probes for research purposes or as diagnostic reagents even though such fragments may show little or no blood clotting activity.
  • Such proteins or polypeptides preferably comprise at least 50 amino acids, more preferably at least 100 amino acids.
  • Preferred domains, epitopes and fragments of Factor VIII and Factor VIII -like proteins include the light chain thereof, parts of the light chain containing the domains A3-C1, C1-C2, A3, Cl, or C2 and the individual domains A3, Cl and C2.
  • Factor VIII and Factor Vlll-like proteins that can be purified according to the present invention also include all recombinant proteins, hybrids, derivatives, mutants, domains, fragments, and epitopes described in US 7,122,634, US 7,041,635, US 7,012,132, and US 6,866,848, all of which are incorporated herein by reference in their entirety.
  • amino acid encompasses any organic compound comprising at least one amino group and at least one acidic group.
  • the amino acid can be a naturally occurring compound or be of synthetic origin.
  • the amino acid contains at least one primary amino group and/or at least one carboxylic acid group.
  • amino acid also refers to residues contained in larger molecules such as peptides and proteins, which are derived from such amino acid compounds and which are bonded to the adjacent residues by means of peptide bonds or peptido-mimetic bonds.
  • the invention relates to compounds comprising peptides and peptide derivatives of formula 1
  • B is a tetrapeptide, pentapeptide, hexapeptide or heptapeptide binding to FVIII and/or FVIII-like proteins or domains thereof, Q is absent or an organic spacer molecule and
  • X is absent or an organic anchoring molecule, as well as their pharmaceutically acceptable salts.
  • the peptides according to the present invention bind with high affinity towards FVIII and/or FVIII-Hke proteins or domains thereof.
  • Affinity is the force of attraction between atoms or molecules that helps to keep them in combination. This is the basis for affinity chromatography. Affinity can also be defined as a measure of the intrinsic binding strength of the ligand binding reaction. The intrinsic attractiveness of the binder for the ligand is typically expressed as the equilibrium constant (Ka) of the reaction.
  • the equilibrium constant Ka [Ligand-Binder]/[Ligand][Binder], where [ ] represents the molar concentration of the material at equilibrium,
  • a peptide or peptidomimetic is considered to show affinity to FVIlI or FVIII-like proteins if a binding to FVIII is measured according to the test protocol below, which is at least 10%, preferably at least 25% and most preferably at least 40%.
  • the degree of binding is measured by reproducing the experiment described in Example 1 below using I-labeled FVIII.
  • the peptide or peptide derivative B of the present invention will be chemically bound to the surface of the preferred embodied solid carrier matrix, preferably, with the help of an anchorage molecule X and/or a spacer molecule Q or, if Q and X are missing, preferably by a SH, N 3 , NH-NH 2 , 0-NH 2 , NH 2 , -CH 2 -L, C ⁇ CH, carbonyl or carboxyl group of the compound B.
  • L comprises a leaving group, like Cl, Br or I.
  • chemical binding' 1 includes covalent, ionic, hydrophobic and/or other complex interactions, as well as mixtures and combinations thereof, between two (o ⁇ more) atoms, or one (or more) atom(s) and one (or more) compound(s), or, two (or more) compounds.
  • B is E 1 HN-(Z) n -COE 2 wherein
  • E is H, R 1 , -COR 1 or -CO 2 R 1 , is -OR 2 or -NHR 2 ,or -NH-NHR 2 wherein
  • R 1 is Cl-4 alkyl, Ar or CH 2 -Ar, R 2 is H or R 1 ,
  • Ar is an unsubstituted phenyl; or is a substituted phenyl which is one-, two-, or threefold substituted with A, OH, OA, CF 3 , OCF 3 , CN, NO 2 or Hal, which can be substituted one-, two-, or threefold with an A, OH, OA, CF 3 , OCF 3 , CN, NO 2 or Hal substituted phenyl, in such a way that an unsubstituted or substituted bipheny] is created; or is Het, wherein Hal is selected from F, Cl, Br or I, Het is a saturated or unsaturated mono- or bicyclic heterocyclic residue with 5 to 12 ring members, comprising 1 to 3 N- and/or 1 S- or O- atoms, wherein the heterocyclic residue can be substituted one- or two-fold with CN 5 Hal, OH , NH 2 , COOH, OA, CF 3 , A, NO 2 , Ar or O
  • A is COOH, NH 2 or alkyl with 1-6 C-atoms, unsubstituted or substituted with COOH or NH 2
  • Z is a naturally occurring or non-proteino genie amino acid residue or derivative thereof
  • n is an integer between 4 and 7, wherein
  • w is an integer from between 1 to 8
  • x is an integer from between 1 to 5
  • y is an integer from between 1 to 6
  • z is an integer from between 1 to 6.
  • Organic spacer molecules are known per se, "Organic” refers to all carbon compounds except carbide arid carbonate compounds, see also Beilstein's Handbook of Organic Chemistry.
  • the organic spacer molecule is a linear hydrocarbon having a functional groups at one or both terminal ends. The hydrocarbon chain can be modified.
  • X is an organic anchoring molecule selected from one of the following groups a naturally occurring or non-proteino genie amino acid,
  • a 1 is NR 2 , CO, CHR 2 , O, or S,
  • a 2 is SH, N 3 , NH ⁇ NH 2 , 0-NH 2 , NH 2 , Hal ! , C ⁇ Cti, CR 6 O, or carboxyl,
  • R 2 is as defined before
  • R 4 is H, R 6 , -COR 6 , -COOR 6 ,
  • R 5 is -OR 6 or -NHR 4 ,
  • R 6 is H, or is unsubstituted phenyl or with A, OH 5 OA,
  • Hal is F, Cl, Br or I
  • Hal 5 is Cl, Br or I
  • Anchoring molecules are molecules or molecule-groups which can be applied for linking fragments (i.e. a compound and a resin). Such anchoring molecules are known per se. Usually anchoring molecules comprise two or more functional groups which can form a chemical binding.
  • Another embodiment is characterized in that at least one residue Z from (Z) n with n between 4 to 7 is selected from alanine, valine, serine, threonine or ⁇ -aminobutyric acid side chain.
  • a further embodiment is characterized in that at least one residue Z from (Z) 11 with n between 4 to 7 is selected from cysteine, /r ⁇ mo-cysteine or D-cysteine.
  • Another embodiment is characterized in that at least one residue Z from (Z) n with n between 4 to 7 is selected from glutamic or aspartic side chain.
  • Another embodiment is further characterized in that at least one residue Z from (Z) n with n between 4 to 7 is selected from phenylalanine, tyrosine, O-methylated tyrosine, I- naphthylalanine, 2-naphthylaianine, tryptophane, p-benzoylphenyialanine or -CH 2 -Ar side chain, wherein Ar is as defined above.
  • An embodiment is further characterized in that E 1 is H or acetyl.
  • Another embodiment is further characterized in that E 2 is OH or NH 2 .
  • Another embodiment is further characterized in that Q is [ ⁇ NH-(CH2) X -CO] W with x and w are as defined before.
  • An embodiment of the invention comprises compounds further characterized in that x is 1 or 5 and w is 0 or 1.
  • X is -A'-CHCCORVCCHaVA 2 wherein
  • a 1 is NH
  • R 5 is OH or NH 2
  • a 2 is SH
  • a preferred embodiment of the invention comprises compounds further characterized in that q is l .
  • Another embodiment of the invention comprises compounds further characterized in that wherein A 1 is CO R 4 is H
  • a 2 is SH and q is as defined before.
  • a preferred embodiment of the invention comprises compounds further characterized in thai q is l .
  • amino acid residues can be independently chosen from L- or D- ⁇ -amino carbonic acid residues, ⁇ -amino carbonic acid residues, aza-amino carbonic acid residues and peptoid- amino carbonic acid residues.
  • a preferred embodiment of the invention comprises compounds further characterized in that the amino acid residues are chosen from ⁇ -amino acid residues.
  • Another embodiment comprises compounds further characterized in that at least two of the residues Z are bound by acid-amide bonds -CO-NH- or -NH-CO-.
  • a more preferred embodiment of the invention comprises compounds further characterized in that at least two of the residues Z are bound by N -methylated acid-amide bonds -CO-NCH 3 - or -NCH 3 -CO-.
  • Another embodiment of the invention comprises compounds further characterized in that one or more peptide bonds can be independently modified.
  • a further embodiment of the invention comprises compounds further characterized in that at least two of the residues Z are bound by -CH 2 -NH- or -NH-CH 2 - bonds.
  • Another embodiment of the invention comprises compounds further characterized in that the direction of the peptide sequence is inverted (“retropeptide").
  • Another embodiment of the invention comprises compounds further characterized in that the direction of the peptide sequence is inverted and the amino acid residues are chosen from D- ⁇ - amino acid residues ("retro-inverso-peptide").
  • the following Embodiments AA and BB are of particular interest:
  • Embodiment AA 5 B is preferably represented by the general formula
  • E 1 and E 2 are as defined above, and
  • Zl is a naturally occurring or non-proteinogenic amino acid residue or a derivative thereof, or Zl may be absent.
  • Zl is an aromatic naturally occurring or non-proteinogenic amino acid residue or a derivative thereof. More preferred groups Zl include proteinogenic aromatic amino acids (Phe, Tyr, Trp, His) and derivatives thereof, in particular those derivatives carrying one to three substituents selected independently from Cj diligent4 alkyl groups, halogen atoms or benzyl groups at the side chain thereof. Typical examples of such derivatives are Tyr(OMe), Tyr(OBn), Trp(Me).
  • Zl that are more preferred include cyclohexylalanine, 1-naphthylalanine, 2-naphthylalanine, thienylalanine, benzothienylalanine, phenylglycine, p-benzoylphenylalanine, homophenylalanine, homotyrosine, homotryptophane, homohistidine and their derivatives as described above with respect to the natural amino acids.
  • Particularly preferred groups Zl are unsubstituted Phe, Phe carrying one or two subsiituents independently selected from C 1.4 -alkyl and O-C]_4-alkyl as well as Tyr or Tyr substituted with one or two substituents independently selected from C1..4- alkyl and O-Ci_4-alkyl. Most preferred groups Zl are selected from Phe, Tyr and Tyr(OMe).
  • E'-HN- may be absent and Zl can be represented by a residue of the formula
  • a ⁇ represents NR 21 , CO, CHR 2 *, O 3 or S, and forms a peptide bond or peptidomimetic bond with the adjacent residue
  • R1 1 represents C 1,4 alkyi, phenyl or benzyl, and N(R ⁇ ) 2 , wherein the alkyl, phenyl or benzyl group carries at least one group N(R 2 ⁇ ) 2 and optionally one or more substituents independently selected from A a and
  • N(R 2 ⁇ ) 2 wherein two or more A a 's and/or two or more R 2 ⁇ 's may be the same or different from each other, and
  • Ar a is an aromatic group having a mono-, bi- or tricyclic aromatic ring system with 6 to 14 carbon atoms, a saturated or partially unsaturated C5_]4 mono- or bicyclic alkyl group, each of which may be unsubstituted or one-, two-, or threefold substituted with group independently selected from A a , O-Ar 11 , C(O)-Ar 1 ! , CH 2 -Ar" > OH,
  • Hal is selected from F, Cl, Br or I,
  • Het a is a heterocyclic residue as defined above with respect to ZA,
  • a a represents COOR 2 I, N(R 2 ⁇ ) 2 or a linear, branched or cyclic alkyl group with 1-6 C-atoms, which may be unsubstituted or be substituted with COOR 2 or N(R 2 ) 2 ml and ol are independently selected from O, 1, 2, 3 and 4,
  • nl 0 or 1
  • R ⁇ ⁇ is H, C].4 alkyl, phenyl or benzyl or, in the case of peptoid-amino acids, the amino acid side chain.
  • residues Zl of the above formula (II- 1) wherein Ar a represents phenyl, 2-hydroxyphenyi, 3-hydroxyphenyl, 4-hydroxyphenyl, 1-naphthyl, 2-naphthyl, p- benzoylphenyl, biphenyl, 2-indolyl, 3-indolyl, thiophene, benzothiphene, each of which may carry one to three substituents independently selected from A a and Hal, and wherein all other groups are as defined above.
  • Ar a represents phenyl, 2-hydroxyphenyi, 3-hydroxyphenyl, 4-hydroxyphenyl, 1-naphthyl, 2-naphthyl, p- benzoylphenyl, biphenyl, 2-indolyl, 3-indolyl, thiophene, benzothiphene, each of which may carry one to three substituents independently selected from A a and Hal, and wherein all other groups are as defined above.
  • Z2 is a naturally occurring or non-proteino genie amino acid residue or a derivative thereof.
  • Z2 represents Cys, D-Cys or homo-Cys and Q and X are absent.
  • Z3 is a naturally occurring or non-proteinogenic amino acid residue or a derivative thereof.
  • Z3 is a naturally occurring or non-proteinogenic amino acid residue or a derivative thereof, selected from GIy, Ala, Ser, Thr, VaI, and Abu ( ⁇ -aminobutyric acid).
  • Z4 is a naturally occurring or non-proteinogenic amino acid residue or a derivative thereof with a large side chain.
  • the side chain comprises at least 3 carbon atoms, preferably at least 5 carbon atoms and more preferably from 6 to 25 carbon atoms. One or more of these carbon atoms may be replaced by a heteroatom selected from N, O and S.
  • the side chain of Z4 contains preferably a cyclic group, which may be monocyclic, bicyclic or tricyclic. Moreover, this cyclic group may be saturated, unsaturated or aromatic. Aromatic groups are more preferred, as weli as bicyclic groups. Aromatic bicyclic groups are particularly preferred.
  • bodiment Z4 may also preferably be a residue of the formula Ar b - ⁇ CH2) m4 - ⁇ CHA I4 ), 14 -(CH 2 ) o 4-A 14 (IM) wherein
  • a ⁇ 4 both represent a group independently selected from NR 24 , CO, OCO,
  • ⁇ r is an aromatic group having a mono-, bi- or tricyclic aromatic ring system with 6 to 14 carbon atoms, a saturated or partially unsaturated C5-14 mono- or bicyclic alkyl group, each of which may be unsubstituted or carry one to three substituents independently selected from A b , Ar* 4 , O ⁇ Ar 14 , C(O)-Ar * 4 , CH 2 -ArI 4 , OH, OA ⁇ CF 3 , OCF3, CN, NO2, Hal; or Ar may be He ⁇
  • Hal is selected from F, Cl, Br or I,
  • Ar ⁇ 4 is an aromatic group having a mono-, bi- or tricyclic aromatic ring system with 6 to 14 carbon atoms, preferably a phenyl group or a naphthyl group, more preferably a phenyl group.
  • Ar ⁇ 4 may itself be unsubstituted or carry one to three substituents independently selected from A b , OH, 0A b , CF 3 , OCF 3 , CN, NO 2 , and Hal;
  • Het represents a saturated, partially or completely unsaturated mono- or bicyclic heterocyclic residue with 5 to 12 ring members, comprising 1 to 3 N- and/or 1 S- or O- atoms.
  • heterocycles on which the heteroaryl radical or the radical of the monocyclic or bicyclic 5-membered to 12-membered heterocyclic ring can be based are pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, tetrazole, pyridine, pyrazine, pyrimidine, indole, isoindole, indazole, phthalazine, quinoline, isoquinoline, quinoxaline, quinazoline, cinnoline, D- carboline or benz ⁇ -fused, cyclopenta-fused, cyclohexa-fused or cyclohepta-fused derivatives of these heterocycles.
  • Nitrogen heterocycles can also be present as N-oxides.
  • Radicals which can be heteroaryl or the radical of a monocyclic or bicyclic 5-membered to 12-membered heterocyclic ring are, for example, 2- or 3-pyrrolyl, phenylpyrrolyl, for example 4- or 5-phenyl- 2-pyrroly ⁇ , 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 4-imidazolyl, methylimidazolyl, for example l-methyl-2-, -4- or -5-imidazolyl, 1,3- thiazol-2-yl 5 2-pyridyl, 3-pyridyl, 4-pyridyl, N-oxido-2-, -3- or -A- pyridyl, 2-pyrazinyl, 2-, 4- or 5-pyrimidinyl, 2-, 3- or 5-indolyl, substituted 2-indolyl, for example 1 -methyl-, 5 -methyl-, 5-methoxy-, 5- benzyloxy-, 5-
  • Heterocyclic radicals representing the radical Het can be unsubstituted on carbon atoms and/or ring nitrogen atoms or mono substituted or polysubstituted, for example disubstituted, trisubstituted, tetrasubstit ⁇ ted or pentasubstituted, by identical or different substituents.
  • Carbon atoms can be substituted, for example, by (Cj-
  • Cg)-alkyl, in particular (Cj-C4)-alkyl, (Cj-Cj ⁇ -alkoxy, in particular (C ⁇ -C4)-allcoxy (the alkyl moiety of the aforementioned substituents may itself be unsubstituted or substituted with COOR 24 or N(R 24 )2, halogen, nitro, N(R ⁇ ) 2 , trifluoromethyl, OCF3, hydroxy!, oxo, cyano, COOR24 ; aminocarbonyl, (Ci-C4)-aIkoxycarbonyl, phenyl, phenoxy, benzyl, benzyloxy, tetrazolyl, in particular by (Cj-C ⁇ -alkyl, for example methyl, ethyl or tert-butyl, (Cj-C_i)-alkoxy : for example methoxy, hydroxy!, oxo, phenyl, phenoxy, benzyl, benzyJ
  • Sulfur atoms can be oxidized to the sulfoxide or to the sulfone.
  • the radical Het are 1-pyrrolidinyl, 1-piperidinyl, 1 -piper azinyl, 4- substituted 1 -piperazinyl, 4-morpholinyl, 4-thiomorpholinyI, l-oxo-4- thiomorpholinyl, 1 , 1 -dioxo-4-thiomorpholinyl, perhydroazepin-1 -yl, 2,6-dimelhyl- 1-piperidinyl, 3,3-dimethyl-4-morphoIinyl, 4-isopropyi- 2,2,6,6-tetramethyl-l -piperazinyl, 4-acetyl-l -piperazinyl, and 4- ethoxycarbonyl- 1 -piperazinyl.
  • A represents COOR24 ; N(R ⁇ ) 2 or a linear, branched or cyclic alkyl group with 1-6 C-aloms, which may be unsubstituted or be substituted with COOR 24 or N(R24) 2 ,
  • m4 and o4 are independently selected from 0, 1, 2, 3 and 4,
  • n4 is 1 .
  • R24 is H, C].4 alkyl, phenyl or benzyl or, in the case of peptoid-amino acids, the amino acid side chain.
  • More preferred groups Z4 include proteinogenic aromatic amino acids (Phe, Tyr, Trp, His) and derivatives thereof, in particular those derivatives carrying one to three substituents selected independently from R°4 (as defined below) at the side chain thereof.
  • Typical examples of such derivatives are Tyr(OMe), Tyr(OBn), Trp(Me), More preferred groups Z4 include derivatives of Tyr, Tyr(OMe) and Tyr(OBn), wherein the substituent is attached to the meta- or otho-position of the phenyl group.
  • Further groups Z4 that are more preferred include cyclohexylalanine, 1- naphthylalanine, 2-naphthylalanine, 2-tbienylalanine, 3-thienylalanine, benzothienylalanine (wherein the bicyclic ring system can be attached to the remainder of the molecule at any position of the ring system, preferably at the 2- or 3-position of the thienyl ring), phenylglycine, p-benzoylphenylalanine, homophenyialanine, homotyrosine, homotryptophane, homohistidine and their derivatives as described above with respect to the natural amino acids.
  • Ar represents a preferred subgroup of the aromatic groups defined by Ar , including phenyl, 2-hydroxyphenyl, 3-hydroxyphenyl, 4- hydroxyphenyl, 1-naphthyl, 2-naphthyl, p-benzoylphenyl, (ortho-, meta-, or para-)biphenyl, 2-indolyl, 3-indolyI, 2-thiophenyl, 3- thiopheny], 2-benzothiphenyl, 3-benzothiophenyI, each of which may b carry one to three substituents independently selected from A and Hal, and
  • R.64 represents H, Cj .4 alkyl, phenyl or benzyl, each of which may be unsubstituted or one-, two-, or threefold independently substituted with A b , OH, OA b , CF 3 , OCF 3 , CN 5 NO 2 or HaI.
  • Particularly preferred groups Z4 are selected from Trp, 1-naphthyl alanine, 2- naphthylalanine, p-benzoylphenylalanine, benzothienylalanine, and groups of the
  • Z4 that are selected from Trp and p-benzoylphenylalanine.
  • Z5 is missing or is a naturally occurring or non-proteinogenic amino acid residue or a derivative thereof.
  • Preferably Z5 is not aromatic.
  • Z5 is a polar amino acid including Ser, Thr, GIu, Asp, Asn, GIn, Arg, Lys, and derivatives thereof (including, for instance N-alkylated and Ca- methylated polar amino acids and polar amino acid derivatives with a modified side chain length such as homo-derivatives and Orn).
  • Particularly preferred groups Z5 are selected from polar amino acids as defined above, which carry a negative charge under physiological conditions, such as GIu, Asp, homo-Glu and homo- Asp. Most preferred groups Z5 are GIu or Asp.
  • Z6 is a residue as defined above for Zl . That is, Z6 is a naturally occurring or non- proteinogenic amino acid residue or a derivative thereof or a group represented by genera] formula (II-6),
  • a 16 represents NR 26 , CO, CHR 26 , O, or S, and forms a peptide bond or peptidomimetic bond with the adjacent residue
  • R*6 represents C ⁇ . ⁇ . alkyl, phenyl or benzyl, and N(R 2 ⁇ ) 2 , wherein the alkyl, phenyl or benzyl group carries at least one group N(R 2 ⁇ ) 2 and optionally one or more substituents independently selected from A and N(R 2 ")2, wherein two or more A 's and/or two or more R 2 "'s may be the same or different from each other, or R !6 represents A 16 in case that Z7 is not absent, and
  • Ar is an aromatic group having a mono-, bi- or tricyclic aromatic ring system with 6 to 14 carbon atoms, a saturated or partially unsaturated C5_i4 mono- or bicyclic alkyl group, each of which may be unsubstituted or one-, two-, or threefold substituted with group independently selected from A C 3 O-Ar 16 , C(O)-Ar 16 , CH 2 -Ar 16 , OH, OA 0 , CF 3 , OCF 3 , CN, NO 2 or HaI 5 or He/,
  • Hal is selected from F, Cl, Br or I,
  • Het is a heterocyclic residue as defined above with respect to Z4,
  • A represents COOR ⁇ 6 , N(R 2 ⁇ ) 2 O r a linear, branched or cyclic alkyl group with 1-6 C-atoms, which may be unsubstituted or be substituted with COOR 26 or N(R26) 2
  • m6 and 06 are independently selected from O, 1 , 2, 3 and 4,
  • n6 is O or 1 , with the provisos that -COE 2 is absent if Z7 is absent and that no is 1 if Z7 is not absent, and
  • R26 is H, C].4 alkyl, phenyl or benzyl or, in the case of peptoid-amino acids, the amino acid side chain.
  • More preferred groups Z6 include proteinogcnic aromatic amino acids (Phe, Tyr, Trp, His) and derivatives thereof, in particular those derivatives carrying one to three substituents selected independently from C ⁇ .4 alkyl groups, halogen atoms or benzyl groups at the side chain thereof.
  • Typical examples of such derivatives are Tyr(OMe), Tyr(OBn), Trp(Me).
  • Z6 that are more preferred include cyclohexylalanine, 1-naphthyl alanine, 2-naphthylalanine, thienylalaninc, benzothienylalanine, phenylglycine, p-benzoylphenyl alanine, homophenylalanine, homotyrosine, homotryptophane, homohistidine and their derivatives as described above with respect to the natural amino acids.
  • Z6 include groups represented by the above general formula (III), wherein Ar represents phenyl, 2-hydroxyphenyl, 3-hydroxyphenyl, 4- hydroxyphenyl, 1-naphthyl, 2-naphthyl. p-benzoylphenyl, biphenyl, 2-indolyl, 3- indolyl, thiophene, benzotbiphene, each of which may cany one to three substituents independently selected from A and HaL and wherein the remaining substituents of formula (III) are as defined above with respect to Z4.
  • Ar represents phenyl, 2-hydroxyphenyl, 3-hydroxyphenyl, 4- hydroxyphenyl, 1-naphthyl, 2-naphthyl.
  • Z6 are selected from 1-Nal, Phe, Tyr and Tyr(OMe),
  • residues Z4 and Z6 or Z4 and Z5 as well as 25 and Z6 in the respective dipeptide and tripeptide residues of B of the present invention are each linked via a peptide bond or peptidomimetic bond.
  • Preferred compounds of the present invention include those peptide groups B that contain a tripeptide moiety selected from the following combinations of residues:
  • Z7 is a natural occurring or non-pro teinogen ⁇ c amino acid residue or a derivative thereof, or Z7 may be absent. Preferably, Z7 is absent.
  • zl to z7 are the D-enantiomers of the residues Zl to Z7 defined above.
  • the compounds of this Embodiment BB represent the "'retro-inverso-peptide" derivatives of the compounds according to the preceding embodiment.
  • Preferred compounds of Embodiment AA are the above-mentioned compounds of sequences S 1-S7, S9-S12, S14-S16, S18, S20, S24-S26, S40, S69, S70 and S80-S82.
  • Preferred compounds of Embodiment BB are the above-mentioned compounds of sequences S8, S13, S17, S19 5 S83 and S 84.
  • Another embodiment of the invention comprises a compound for the treatment of diseases, wherein said compound is selected from the compounds and preferred compounds disclosed above.
  • the compounds and preferred compounds disclosed above can be bound to a solid carrier matrix with a surface
  • the solid carrier matrix to which, according to an embodiment of the invention, the above compounds are bound can comprise inorganic or organic, especially polymeric material.
  • the compounds and preferred compounds disclosed above are bound to a solid carrier matrix wherein the compound or the compounds are chemically bound to the surface of the solid carrier matrix, for example to a resin.
  • the compounds and preferred compounds disclosed above are bound to a solid carrier matrix wherein the compound or the compounds are bound through organic spacers to the surface of the solid carrier matrix, for example to a resin.
  • the compounds and preferred compounds disclosed above are bound to a solid carrier matrix wherein the compound or the compounds are bound through an organic anchoring molecule to the surface of the solid carrier matrix, for example to a resin.
  • the compounds and preferred compounds disclosed above are bound to a solid carrier matrix wherein the compound or the compounds are bound through organic spacers and an organic anchoring molecule to the surface of the solid carrier matrix, for example to a resin.
  • the solid carrier material comprises inorganic or organic, especially polymeric, material. Therefore, the same polymeric material (i.e. linear polysaccharide) can be utilized which is usually employed for the chromatography of biopolymers.
  • polymers exerting a hydrophilic surface are suitable as a chromatography solid carrier material, i.e. a resin.
  • a resin for example, the Toyopearl AF-Epoxy-650M resin is employed.
  • Such solid carrier material can also be provided with an additional anchoring molecule offering, for example, a SH, N 3 , NH-NH 2 , 0-NH 2 , NH 2 , -CH 2 -L, O ⁇ CH, epoxy, carbonyl or carboxyl group for immobilization of compounds like peptides and peptide derivatives according to formula I.
  • an additional anchoring molecule offering, for example, a SH, N 3 , NH-NH 2 , 0-NH 2 , NH 2 , -CH 2 -L, O ⁇ CH, epoxy, carbonyl or carboxyl group for immobilization of compounds like peptides and peptide derivatives according to formula I.
  • the preferred compounds of the current invention interact with FVIII, FVIII-like proteins and/or domains thereof
  • the preferred compounds comprising peptides and peptide derivatives are suitable for diagnostic devices and kits.
  • the preferred diagnostic device or kit comprises at least one compound, having a high affinity for FVIII or FVIII-like proteins or domains thereof, a solid carrier matrix to which at least one compound may optionally be bound chemically, and other reagents, if needed.
  • the compound is labeled.
  • the compound i.e. by using radioactive markers, by using fluorescent ligands, by using the avidine/streptavidine system, or, as is common in the ELISA technique, by using enzymes which provoke color reactions.
  • the invention comprises further a method of detecting, identifying, diagnosing, isolating, purifying, stabilizing and/or enhancing the activity of FVIII or FVIII-Hke proteins or domains thereof comprising contacting a sample comprising a FVIII or FVIII-Hke protein or domains thereof with a matrix comprising an immobilized compound, under conditions suitable for binding between FVIIl or FVIII-Uke protein or domains thereof and said compound.
  • the compound is immobilized on a solid carrier and the solid carrier is a polymeric material
  • the compound is selected from the group of Sl, S2, S3, S4, S5, So, S 7, S8, S9, SlO, SI l , S12, S13, S14, S15, S16, S17 5 S18, S19, S20, S21, S22, S23, S24, S25, S26, S27, S28, S29, S30, S31 5 S32, S33, S34, S35, S36, S37, S38, S39, S40, S41, S42, S43, S44, S45, S46, S47, S48, S49, S50, S51, S52, S53, S54, S55, S56, S57, S58, S59 : S60, S61, S62, S63, S64, S65, S66, S67, S68, S69, S70, S71 , S72, S73, S74, S75, S76, S77, S78 or S79, as defined above.
  • the polymeric material is a resin.
  • the sample is a cell culture supernatant.
  • the sample is a body fluid.
  • the body fluid comprises serum or whole blood.
  • Another embodiment of the invention comprises a method for producing a FVIII or FVIII-Hke protein or domains thereof containing a pharmaceutical composition or medicament, comprising a method for purifying a FVIII or FVIII-Hke protein or domains thereof and formulating the purified FVIII or FVIII-like protein or domains thereof.
  • Yet another embodiment of the invention comprises a method for producing and/or stabilizing a FVIII or FVIII-like protein or domains thereof containing a pharmaceutical composition or medicament, comprising a method for purifying and/or stabilizing a FVIII or FVIII-like protein or domains thereof and formulating said purified and/or stabilized FVIII or FVIII-like protein or domains thereof.
  • Another embodiment of the invention comprises a method for enhancing the activity of a FVIII or FVIII-like protein or their domain containing a pharmaceutical composition or medicament, comprising a method for enhancing the activity of a FVIII or FVIII-like protein or domains thereof and formulating said activity-enhanced FVIII or FVII ⁇ -like protein or domains thereof.
  • the compound is used for labeling, detecting, diagnosing, monitoring, identifying, isolating, purifying, stabilizing and enhancing the activity of a FVlII or FVIII-like protein or domains thereof.
  • the compound is labeled.
  • the compound i.e. by using radioactive markers, by using fluorescent ligands, by using the avidine/streptavidine system, or, as is common in the ELISA technique, by using enzymes which provoke color reactions.
  • the molecules of the present invention relates to compounds comprising peptides and peptide derivatives which are suitable for labeling, detecting, identifying, isolating, purifying, stabilizing and/or enhancing the activity of FVIII and/or FVIII-like proteins and/or domains thereof.
  • Preliminary experiments in the context of the present invention showed that the addition of at least one of the peptides according to the invention leads to a stabilization of the activity of FVIII and/or FVIII-like proteins and/or domains thereof or enhancement of the activity of FVIII and/or FVIII-like proteins and/or domains thereof in a standard chromogenic assay by 10%, preferably by 30%.
  • building blocks of the compounds of the formula I i.e. the amino acids mentioned above
  • the building blocks of the compounds of the formula I can occur in a plurality of enantiomeric forms, all these forms and also mixtures thereof (for example the DL forms) are included.
  • amino acids may, for example, as a constituent of compounds of the formula I, be provided with corresponding protecting groups known per se.
  • protecting groups are derivatives of Asp and GIu, particularly methyl-, ethyl-, propyl-, butyl-, ter ⁇ -bntyl-, neopentyl- or benzylester of the side chain or derivatives of Tyr, particularly methyl-, ethyl-, propyl-, butyl-, tert-butyl-, neopentyl- or benzylethers of the side chain.
  • the compounds may furthermore carry one or more of the additional protecting groups that are described below in connection with the preparation of the compounds of the present invention.
  • iV-terminal modified or carboxy-terminal modified derivatives are part of this invention.
  • Favored groups are amino-terminal methyl-, ethyl-, propyl-, butyl-, tert-butyl-, neopentyl-, phenyl- or benzyl-groups, amino-terminal groups like BOC, Mtr, CBZ, Fmoc, and, particularly, acetyl, benzoyl or (indol-3-yl)carbonic acid groups, furthermore, carboxy-terminal methyl-, ethyl-, propyl, butyl-, terl-butyl-, neopentyl- or benzylester, methyl-, ethyl-, propyl-, butyl-, tert-butyl-, neopentyl- or benzylamides and, particularly, carboxamides.
  • Alpha amino groups may be protected by a suitable protecting group selected from aromatic urethane-type protecting groups, such as allyloxycarbonyl, benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl, such as p-chlorobenzyloxycarbonyl, p- nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-biphenyl-isopropyloxycarbonyl, 9- fluorenylmethyloxycarbonyl (Fmoc) and p-methoxybenzyloxycarbony ⁇ (Moz); aliphatic urethane-type protecting groups, such as t-butyloxycarbonyl (Boc), diisopropylmethyloxycarbonyl, and isopropyloxycarbonyl.
  • Fmoc is most preferred for alpha amino protection.
  • Amino acids which can be used for the formation of the peptides and peptide derivatives according to the present invention, can belong to both natural occurring and non- proteinogenic amino acids.
  • Amino acids and amino acid residues can be derivatized, whereas TV-methyl-, TV-ethyl-, ⁇ L propyl- or TV-benzyl- derivatives are favored.
  • TV-alkylation of the amide binding can have a strong influence on the activity of the corresponding compound (Levian-Teitelbaum, D.; Kolodny, N.; Chorev, M.; Selinger, Z.; Gilon, C.
  • amino acids that can be used include amino acids with modifications in the side chain, /?-amino acids, aza-amino acids (derivatives of ⁇ -amino acids, where the ⁇ -CH-group is substituted by a N-atotn) and/or peptoid-amino acids (derivatives of ⁇ -amino acids, where the amino acid side chain is bound to the amino group instead to the ⁇ -C-atom) or cyclised derivatives from the above mentioned modifications.
  • amino acids with modifications in the side chain include amino acids with modifications in the side chain, /?-amino acids, aza-amino acids (derivatives of ⁇ -amino acids, where the ⁇ -CH-group is substituted by a N-atotn) and/or peptoid-amino acids (derivatives of ⁇ -amino acids, where the amino acid side chain is bound to the amino group instead to the ⁇ -C-atom) or cyclised derivatives
  • homo- derivatives of naturally occurring amino acids as building blocks. These are derivatives of the naturally occurring amino acids, wherein a methylene group is inserted into the side chain, immediately adjacent to Ca. Similarly, it is possible to use ⁇ -methylated derivatives of naturally occurring amino acids in accordance with the present invention.
  • the inventive compounds are peptido- mimetics.
  • peptido-mimetic comprises compounds containing non-peptidic structural elements which are capable of mimicking or antagonizing the biological action(s) of a parent peptide. Such compounds preferentially comprise few (or no) peptide bonds.
  • substituent R 21 may be the side chain of the respective amino acid (peptoid amino acid). In this case, the adjacent Ca does not carry the side chain.
  • Other substituents R 21 are present in addition to the side chain attached to Ca.
  • the individual R 21 's can be the same or different from each other,
  • the invention furthermore relates to the process for the preparation of compounds of the formula I and salts thereof. It is contemplated that structural elements like N-terminal modified or carboxy-terminal modified derivatives are part of this invention.
  • the compounds of formula I can have one or more centers of chirality and can therefore occur in various stereoisomeric forms.
  • the invention relates in particular to the compounds of the formula 1 in which at least one of the said residues is mentioned as preferred.
  • the compounds of formula I can be understood as peptides, non-natural peptides or peptide derivatives and may be partially or completely synthesized, for example using solution or solid state synthesis techniques known in the art (Gysin, B. F.; Merrifield, R. B. J. Am. Chem. Soc, 1972, 94, 3102; or Merrifield, R. B. Angew. Chemie Int. Ed 1985, 24(10), 799-810) applying appropriate amino or carboxy building blocks. A sequential synthesis is contemplated. Other organic synthetic methods may be employed in the synthesis of the compounds according to formula I 5 such as the methods described in the textbook Houben- Weyl Methods in Organic Chemistry, Vol. 22 a - d, Editor-in-Chief: M. Goodman, Thieme Verlag, Stuttgart, 2003.
  • the starting materials can also be formed in situ without isolating them from the reaction mixture, but instead subsequently converting them further into the compounds of the formula I.
  • Suitable inert solvents are, for example, hydrocarbons, such as hexane, petroleum ether, benzene, toluene, or xylene; chlorinated hydrocarbons, such as trichlorethylene, 1,2- dichloroethane, tetrachloromethane, chloroform or dichloromethane; alcohols, such as methanol, ethanol, isopropanol, «-propanol, / ⁇ -butanol or tert-butanol; ethers, such as diethyl ether, diisopropyl ether, tetrahydrofurane (THF) or dioxane; glycol ethers, such as 1,2- dimethoxyethane, acetamide, such as jV-methylpyrroHdone, dimethylacetamide or dimethyl formamide (DMF); nitrites, such as acetonitrile; sulfoxides, such as dimethyl sul
  • the compounds of the formula I can furthermore be obtained by liberation from a functional derivative by solvolysis, such as hydrolysis, or hydrogenolysis.
  • Preferred starting materials for the solvolysis or hydrogenolysis are those having corresponding protected amino and/or hydroxyl groups instead of one or more free amino and/or hydroxyl groups, preferably those which carry an amino-protecting group instead of an H atom bonded to an N atom, for example those which conform to the formula I, but contain an NHR' group (in which R' is an amino-protecting group, for example BOC or CBZ) instead of an NH 2 group.
  • R" is a hydroxyl-protecting group, for example ferf-butyl or benzyl
  • the hydroxyl group covalently bonded to the aromatic ring is protected from transformation by a protecting group.
  • R' is a carboxyl-protecting group, for example tert-buiyl or benzyl
  • the oxygen atom of the carboxyl group is protected from transformation by a protecting group.
  • amino-protecting group is known in general terms and relates to groups which are suitable for protecting (blocking) an amino group against chemical reactions, but are easy to remove. Typical for such groups are, in particular, unsubstituted or substituted acyl, aryl, aralkoxymethyl or aralkyl groups. Because the amino-protecting groups are removed after the desired reaction (or reaction sequence) occurs, their type and size are furthermore not crucial; however, preference is given to those having 1-20, in particular 1-8, carbon atoms.
  • acyl group includes acyl groups derived from aliphatic, araliphatic, aromatic or heterocyclic carboxylic acids or sulfonic acids, and, in particular, alkoxycarbonyl, aryloxycarbonyl and especially aralkoxycarbonyl groups.
  • acyl groups are alkanoyl, such as acetyl, propionyl, butyryl; aralkanoyl such as phenylacetyl; aroyl such as benzoyl oder toluyl; aryloxyalkanoyl such as POA; alkoxycarbonyl such as methoxycarbonyl, ethoxycarbonyl, 2,2,2-trichlorethoxycarbonyl, BOC, 2-iodethoxycarbonyl; aralkyloxycarbonyl such as CBZ ("carbobenzoxy”), 4-methoxybenzyloxycarbonyl, Fmoc; arylsulfonyl such as Mtr, Pbf or Pmc.
  • alkanoyl such as acetyl, propionyl, butyryl
  • aralkanoyl such as phenylacetyl
  • aroyl such as benzoyl oder toluyl
  • Preferred amino-protecting groups are BOC, Mtr, CBZ 5 Fmoc, Benzyl and Acetyl groups.
  • hydroxy! -protecting group is likewise known in general terms and relates to groups which are suitable for protecting a hydroxyl group against chemical reactions, but are easy to remove after the desired chemical reaction has been carried out elsewhere in the molecule. Typical for such groups are the above-mentioned unsubstituted or substituted aryl, aralkyl or acyl groups, furthermore also alkyl groups.
  • the nature and size of the hydroxyi- protecting groups are not crucial since they are removed again after the desired chemical reaction or reaction sequence; preference is given to groups having 1-20, in particular 1-10, carbon atoms. Examples of hydroxyl -protecting groups are, inter alia, benzyl, p-nitrobenzoyl, tert-buty ⁇ and acetyl, where benzyl and ten-butyl are particularly preferred.
  • carboxyl-protecting group is likewise known in general terms and relates to groups which are suitable for protecting a carboxyl group against chemical reactions, but are easy to remove after the desired chemical reaction has been carried out elsewhere in the molecule. Typical for such groups are the above-mentioned unsubstituted or substituted aryl, aralkyl or acyl groups, furthermore also alkyl groups.
  • the nature and size of the hydroxyl- protecting groups are not crucial since they are removed again after the desired chemical reaction or reaction sequence; preference is given to groups having 1-20, in particular 1 -10, carbon atoms.
  • Examples of carboxyl-protecting groups are, inter alia, benzyl, f ⁇ rf-butyl and acetyl, where benzyl and tert-bxxty ⁇ are particularly preferred.
  • the compounds of the formula 1 are liberated from their functional derivatives - depending on the protecting group used - for example using strong acids, advantageously using TFA or perchloric acid, but also strong inorganic acids, such as hydrochloric acid or sulfuric acid, strong organic carboxylic acids, such as trichloroacetic acid, or sulfonic acids, such as benzene sulfonic acid or p-toluenesulfonic acid.
  • strong acids advantageously using TFA or perchloric acid
  • strong inorganic acids such as hydrochloric acid or sulfuric acid, strong organic carboxylic acids, such as trichloroacetic acid, or sulfonic acids, such as benzene sulfonic acid or p-toluenesulfonic acid.
  • strong inorganic acids such as hydrochloric acid or sulfuric acid
  • strong organic carboxylic acids such as trichloroacetic acid
  • sulfonic acids such as benzene s
  • Suitable inert solvents are preferably organic, for example carboxylic acids, such as acetic acid, ethers, such as tetrahydrofurane or dioxane, amides, such as DMF, halogenated hydrocarbons, such as dichloromethane, furthermore also alcohols, such as methanol, ethanol or isopropanol, and water. Mixtures of above-mentioned solvents are furthermore suitable. TFA is preferably used in excess without addition of a further solvent, and perchloric acid is preferably used in the form of a mixture of acetic acid and 70% perchloric acid in the ratio 9: 1.
  • the reaction temperatures for the cleavage are advantageously between about 0° and about 50°, preferably between 15° and 30° (room temperature).
  • the BOC, Obut, Pbf, Pmc and Mtr groups can, for example, preferably be cleaved off using TFA in dichloromethane or using approximately 3 to 5N HCl in dioxane at 15-30°, and the Fmoc group can be cleaved off using an approximately 5 to 50% solution of dimethylamine, diethylami e or piperidine in DMF at 15-30°.
  • the trityl group is employed to protect the amino acids histidine, asparagine, glutamine and cysteine. They are cleaved off using TFA / 10% thiophenol, TFA / anisole, TFA / thioanisoie or TFA/TIPS/H2O, with the trityl group being cleaved off all the said amino acids.
  • Pbf pentamethylbenzofuranyl
  • Hydro genolytically removable protecting groups for example CBZ or benzyl
  • a catalyst for example a noble-metal catalyst, such as palladium, advantageously on a solid carrier, such as carbon.
  • Suitable solvents are those indicated above, in particular, for example, alcohols, such as methanol or ethanol, or amides, such as DMF.
  • the hydrogenolysis is generally carried out at temperatures between 0° and 100° and pressures between 1 and 200 bar, preferably at 10-30° and 1-10 bar. Hydrogenolysis of the CBZ group succeeds well, for example, on 5 to 10% Pd/C in methanol or using ammonium formate (instead of hydrogen) on Pd/C in methanol/DMF at 10-30°.
  • a base of the formula I can be converted into the associated acid-addition salt using an acid, for example by reaction of equivalent amounts of the base and the acid in an inert solvent, such as ethanol, followed by evaporation.
  • an acid for example by reaction of equivalent amounts of the base and the acid in an inert solvent, such as ethanol, followed by evaporation.
  • inorganic acids for example sulfuric acid, nitric acid, a hydrohalic acid, such as hydrochloric acid or hydrobromic acid, phosphoric acids, such as orthophosphoric acid or sulfamic acid.
  • Organic acids may be employed including aliphatic, alicyclic, araliphatic, aromatic or heteroaromatic monobasic or polybasic carboxylic, sulfonic or sulfuric acids, for example formic acid, acetic acid, triflouroacetic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, malenic acid, Iacitic acid, tartaric acid, malic acid, citric acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methane- or ethanesulfonic acid, ethanedi sulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenemono- and --disulfonic acids and laurylsulfuric acid. Salts, for example picrates, can
  • an acid of the formula I can be converted into one of its metal of ammonium salts by reaction with a base.
  • Suitable salts are, in particular, the sodium, potassium, magnesium, calcium and ammonium salts, furthermore substituted ammonium salts, for example the dimethyl-, diethyl- or diisopropyl-ammonium salts, monoethanol- diethanol- or diisopropanolylammonium salts, cyclohexyl-, dicyclohexylammonium salts, dibenzyiethylenediammonium salts, furthermore, for example, salts with arginine or lysine.
  • the compounds of the present invention can be used as described below.
  • a definite diagnosis for hemophilia A is evaluated by performing a FVIII assay and measuring the clotting time. Therefore, the patient's plasma is mixed with FVIII-deficient plasma from a patient who congenitaliy lacks FVIII or from an artificially depleted source. The degree of effectiveness in shortening the clotting time will be compared with that of normal plasma.
  • a standard curve is generated using dilutions of pooled fresh normal human plasma with the hemophilic plasma and plotting the clotting times against the dilutions.
  • peptides and peptidomimetics can be replaced by the peptides and peptidomimetics of the present invention.
  • the peptides according to the present invention have major advantages compared to the currently used labile anti- FVIII antibodies employed in ELISA tests.
  • the development of sensitive screening kits for the detection of the total FVIII amount in the patient's plasma permits to benefit from the advantages of the peptides which are found in their greater stability, higher sensitivity and lower assay costs.
  • FVIII shows rapid inactivation. and a short half-life.
  • the half-life of FV1 ⁇ I is defined by the rate of spontaneous dissociation of the A2 subunit from active heterotrimeric FVI ⁇ I (A1/A2/A3-C1-C2) in which the A2 subunit is weakly associated with the Al and the A3-C1-C2 subunits via ionic interactions.
  • the presence of A2 in the heterotrimer is required for normal stability of active FVIIL
  • the peptides and peptidomimetics of the present invention exhibit not only a high affinity to FVIII, but, upon binding, they also serve to stabilize the heterotrimer.
  • a binding of these inventive compounds to FVIII can therefore be used in an advantageous manner in hemophilia A therapy to thereby increase the stability and half-life of FVIII during medical treatment.
  • a longer half-life of FVIII during substitution therapy will ease the patient's well- being as it permits to lower the FVIII infusion frequency.
  • Said stabilization effect may also be used for advantageously increasing the shelf-life of FVIII-containing medicaments prior to their administration.
  • the compounds of the present invention may also carry a marker group such as a radioactive isotope or a functional group that can undergo a colour reaction or the like.
  • a marker group such as a radioactive isotope or a functional group that can undergo a colour reaction or the like.
  • the contacting of such compounds of the present invention with FVIII will lead to the binding of the marker peptide or peptidomimetics to FVIII. This, in turn, permits to detect and, as the case may be, quantify the FVIII present in a sample.
  • the compounds of the present invention may furthermore increase the biological activity of FVIII.
  • the compounds of the present application may have the advantageous effect of inhibiting the binding of antibodies to the administered FVIII.
  • These beneficial effects may be used in therapy by contacting FVIII with a compound of the present invention prior to its administration.
  • the compound of the present invention will bind to FVIII thus forming a complex.
  • Administration of this complex instead of the pure FVIII may lead to an increased biological effect (or, alternatively, permit to administer lower dosages of FVIII).
  • this administration of this complex may be helpful in reducing the deactivating effect of antibodies.
  • the compounds of the present invention may be used for manufacturing a FVIII-based medicament that exhibits higher stability and superior activity as compared with conventional FVIII.
  • Said FVIII-based medicament may also be used for substituting conventional FV ⁇ II in cases where said conventional FVlII is deactivated by antibodies.
  • the present invention also pertains to a method for treating hemophilia A that includes the step of administering an effective dose of said complex of FVIII and the compound of the present invention to a subject in need thereof.
  • Another embodiment of the present invention pertains to the use of the compounds of the present invention for purifying raw FVIII and FVIII-like proteins. This involves preferably the immobilization of the compounds of the present invention on a solid support. More preferably, an affinity chromatography is carried out using a resin coated with the compounds of the present invention.
  • the present invention furthermore pertains to the use of the compounds of the present invention for purifying domains, epitopes and fragments of FVIII and FVIII-like proteins. Whilst such purified domains and the like may not exhibit a clotting activity comparable to FVIII, they may nevertheless be useful in diagnostic kits, as research tools and the like,
  • RT retention time (minutes) on HPLC in the following system:
  • Rink-amid resin stands for 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin, which allows, for example, the synthesis of peptides and peptido mimetic derivatives with C- terminal -CONH 2 groups
  • TCP resin denotes trityl chloride-polystyrene resin.
  • Peptides Sl to S84 were immobilized on the Toyopearl AF-Epoxy-650M resin (Tosoh Biosep) as described by Jungbauer et aL
  • Tosoh Biosep Tosoh Biosep
  • the immobilization buffer 0.2 M sodium bicarbonate. pH 10.3
  • 0.036 g of the dry resin powder corresponding to 0,125 mL of swollen resin
  • i25 l-pd-FVIII Bound/Background ratios were calculated as the amount of 12s I-pd-FVITI, bound to an immobilized peptide, divided by that bound to uncoated control resin, prepared as described above. This ratio represents a Signal/Noise ratio for the micro-beads assay, since ⁇ ⁇ -pd- FVIII bound to peptide represents the signal value and 125 I-pd-FVIII bound to peptide- uncoated resin represents the background (noise) value.
  • Plasma-derived (pd-) human Factor VIII (FVIII) was purified from concentrate by immunoaffinily chromatography on an anli-FVIII monoclonal antibody column followed by subsequent concentration of pd-FVIII by ion-exchange chromatography using Resource Q HR5/5 column. To separate FVIII from vWf concentrate was incubated in 0.35 M NaCl, 0.04 M CaCl 2 , prior to affinity purification.
  • the resin with immobilized peptides was washed in the binding buffer (0.01 M Hepes, 0.1 M NaCl, 5 mM CaCl 2 , 0.01% Tween-80). Subsequently, the resin was diluted in the binding buffer as 1 :7 slurry and aliquoted into Eppendorf tubes (40 ⁇ L per tube). !25 I-pd-FVIII (100000 cpm in 10 ⁇ L) was added to the tubes and the volume of the mixture was adjusted to 100 ⁇ L by adding 50 ⁇ L of the binding buffer containing 4% BSA to give a 2% final concentration of BSA. After 2 hours of incubation at room temperature on a rotator, the samples were washed 4 times in the binding.
  • the compounds Sl to S84 can be synthesized via solid phase peptide synthesis using Fmoc- strategy on TCP resin and on Rink-amide resin respectively (see Fields, G, B.; Nobie, R. L. Int. J. Pept. Protein Res. 199O 5 35, 161).
  • the cleavage of the peptides derivatives from the solid phase and the cleavage of their side chain protection groups can be done simultaneously using 90% TFA 3 5% H 2 O and 5% TIPS.
  • Example 2 Development of novel small peptides (tetrapeptides to hexapeptide) with high affinity to FVIII. Structures comprising C-tenninal cysteine as linker molecule
  • binding molecule is marked in bold, the linker group is marked in italics.
  • Abu a-aminobutyric acid
  • Example 3 Development of novel hexapeptides with high affinity to FVIII, Structures comprising the linker molecule cysteine within the binding sequence
  • binding molecule is marked in bold, the linker group is marked in italics and small letters represent D-amino acids.
  • Novel compounds are furthermore the retro-inverso peptides S8, SIl and S19 which have been derived from the peptides S7, S16 and S18.
  • retro-inverso peptides are synthesized with a reversed sequence, concurrently the configuration of every amino acid is inversed.
  • the resulting compounds S8, S17 and S19 do not contain any proteinogenic amino acid and provide a particularly high protease stability which is crucial for purifying FVIII directly out of protease containing media such as serum or cell culture supernatants.
  • the use of retro- inverso peptides as affinity ligands for FVI ⁇ I has not been described in the literature before.

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Abstract

La présente invention concerne la composition de petites molécules et leur utilisation dans le cadre de l'isolation, la purification, la stabilisation d'une protéine et/ou l'augmentation de son activité. L'invention porte en particulier sur la synthèse et l'optimisation de composés contenant de petits peptides et dérivés de peptides présentant une affinité au facteur de coagulation VIII et/ou aux polypeptides analogues au facteur VIII et/ou à leurs domaines. Ces composés servent à marquer, détecter, identifier, isoler et idéalement purifier, stabiliser et accroître l'activité du facteur VIII, de polypeptides analogues au facteur VIII ou leurs domaines à partir de solutions physiologiques ou non les contenant. De surcroît, ces composés peuvent servir de ligands, liant le facteur VIII, les polypeptides analogues au facteur VIII ou leurs domaines, dans les procédés de la présente invention.
EP07787022A 2006-07-04 2007-07-03 Petits peptides minimisés présentant une forte affinité au facteur viii et protéines similaires au facteur viii Withdrawn EP2046814A2 (fr)

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EP07787022A EP2046814A2 (fr) 2006-07-04 2007-07-03 Petits peptides minimisés présentant une forte affinité au facteur viii et protéines similaires au facteur viii

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EP06013852A EP1876183A1 (fr) 2006-07-04 2006-07-04 Peptides d'une longueur minimalisée ayant une haute affinité pour protéines de facteur VIII et des protéines analogue à facteur VIII
EP07787022A EP2046814A2 (fr) 2006-07-04 2007-07-03 Petits peptides minimisés présentant une forte affinité au facteur viii et protéines similaires au facteur viii
PCT/EP2007/056699 WO2008003707A2 (fr) 2006-07-04 2007-07-03 Petits peptides minimisés présentant une forte affinité au facteur viii et protéines similaires au facteur viii

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JP5946822B2 (ja) 2010-04-21 2016-07-06 ノヴォ ノルディスク アー／エス タンパク質の選択的改変
WO2013098676A1 (fr) * 2011-12-30 2013-07-04 Grifols, S.A. Procédé de purification du facteur viii
PL3628680T3 (pl) 2014-06-12 2022-01-10 Ra Pharmaceuticals, Inc. Modulowanie aktywności dopełniacza
PT3250230T (pt) 2015-01-28 2021-12-07 Ra Pharmaceuticals Inc Moduladores da atividade do complemento
CA3007772A1 (fr) 2015-12-16 2017-06-22 Ra Pharmaceuticals, Inc. Modulateurs de l'activite du complement
CN106046148B (zh) * 2016-06-27 2019-08-23 新乡医学院 一种利用肽配基亲和纯化凝血因子viii的方法
CA3045114A1 (fr) 2016-12-07 2018-06-14 Ra Pharmaceuticals, Inc. Modulateurs de l'activite du complement

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US5994310A (en) * 1998-09-03 1999-11-30 Bayer Corporation Peptide ligands for affinity purification of human Factor VIII
US7112438B2 (en) * 1999-01-04 2006-09-26 Dyax Corp. Binding molecules for human factor VIII and factor VIII-like proteins

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See references of WO2008003707A2 *

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